Core-shell protection type lipid delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicine, and particularly discloses a core-shell protection type intelligent lipid delivery system and a preparation method and application thereof. The structure is as follows: a Y-type DNA scaffold is cross-linked through double-stranded DNA, namely dsDNA, to form a compact net-shaped DNA shell, the compact net-shaped DNA shell is hybridized with DNA on the surface of a cholesterol micelle and is further anchored on the surface of the cholesterol micelle to form a stable core-shell structure, and the surface of the shell is modified and constructed through a nucleic acid aptamer to form a targeted core-shell system. According to the innovative design, through time-space controllable drug release, self-feedback fluorescence monitoring and a cascade amplification drug release mechanism, synergistic interaction of chemotherapy-gene therapy is achieved, and a novel nanotechnology platform is provided for anti-tumor combined therapy.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a core-shell protected intelligent lipid delivery system, its preparation method and application. Background Art

[0002] Cancer treatment has always been one of the major challenges in the medical field. Although traditional chemotherapy methods can inhibit tumor growth to a certain extent, their non-specific mechanism of action often leads to serious toxic and side effects, affecting the normal tissues and organs of patients. In addition, the drug resistance of tumor cells to a single chemotherapy drug has gradually become one of the main reasons for treatment failure. Therefore, it is urgent to develop a cancer treatment strategy that can simultaneously achieve high efficiency and low toxic and side effects.

[0003] In recent years, gene therapy, as an emerging treatment method, has gradually shown its potential in cancer treatment. Small interfering RNA (siRNA) can specifically silence the expression of target genes through the RNA interference mechanism, thereby inhibiting the proliferation and survival of tumor cells. In particular, siRNA targeting Polo-like kinase 1 (PLK1) (siPLK1) has been proven to have significant anti-tumor effects in a variety of tumor models. However, problems such as poor stability, low in vivo delivery efficiency, and lack of targeting of siRNA limit its clinical application.

[0004] At the same time, chemotherapy drugs such as doxorubicin (Dox) still play an important role in cancer treatment. Dox inhibits the activity of topoisomerase II by intercalating into double-stranded DNA, thereby inducing apoptosis of tumor cells. However, side effects such as non-specific distribution and cardiotoxicity of Dox limit its clinical application. Therefore, combining gene therapy with chemotherapy to improve the anti-tumor effect through synergistic effects while reducing toxic and side effects has become a promising treatment strategy.

[0005] The application of nanodelivery systems in cancer treatment provides new ideas for overcoming the limitations of traditional treatments, such as metal nanoparticles, mesoporous silica, graphene, nucleic acid nanostructures, liposomes, polymer nanoparticles, and micelles. These systems can protect drugs from degradation, improve the stability and bioavailability of drugs, and achieve targeted drug delivery. Among them, lipid micelles, as a common nanocarrier, have good biocompatibility and drug encapsulation ability, and can effectively deliver hydrophobic drugs such as Dox. However, a single lipid micelle system still faces problems such as poor stability and insufficient targeting when delivering siRNA. This current situation urgently requires the development of an innovative treatment system with both targeted precision and multi-mechanism synergy.

[0006] In recent years, due to its high programmability and biocompatibility, DNA nanotechnology has gradually become an important part of the nanodelivery system. By rationally designing the DNA structure, efficient encapsulation, targeted delivery, and controlled release of drugs can be achieved. In particular, the Y-shaped scaffold DNA structure, due to its unique spatial configuration and modifiability, can serve as a protective layer for the drug delivery system, significantly improving the stability and targeting of drugs.

[0007] To further improve the accuracy and effectiveness of cancer treatment, the development of intelligent delivery systems has become a research hotspot. Intelligent delivery systems can achieve targeted drug release according to specific stimuli (such as pH, enzymes, miRNAs, etc.) in the tumor microenvironment, thereby maximizing the accumulation of drugs at the tumor site and reducing damage to normal tissues. As an endogenous molecule highly expressed in tumor cells, miRNA has been widely studied as a trigger for drug release. By designing miRNA-responsive DNA structures, specific drug release in tumor cells can be achieved.

[0008] Existing nanodelivery systems have problems such as insufficient targeting, poor stability of nucleic acid drugs, single endogenous stimulus response, low diagnostic and therapeutic synergy, limited gene silencing efficiency, insufficient integration of multimodal therapy, and lack of real-time monitoring ability. The present invention constructs a functionalized core-shell structure APNS to solve the above technical bottlenecks and achieve a new anti-tumor treatment strategy of efficient targeting, stable drug loading, and precise synergistic diagnosis and treatment. Specifically, it includes the following aspects: One is the problem of insufficient targeted delivery efficiency and system toxicity: Existing nanodelivery systems rely on surface charge or simple encapsulation to achieve drug loading, lacking precise targeting, resulting in non-specific distribution and normal tissue toxicity. APNS realizes upright directional targeting through the regular arrangement of AS1411 nucleic acid aptamers hybridized by the Y-shaped scaffold, reducing system toxicity and improving tumor targeting efficiency.

[0009] Two is the problem of poor stability and premature leakage of nucleic acid drugs: Traditional siRNA delivery relies on physical adsorption or electrostatic interaction, and is easily degraded by nucleases or leaked during delivery. APNS encapsulates siRNA in the lipid core through DNA hybridization to form a core-shell protective layer (a reticular DNA dense outer shell + lipid micelle), significantly reducing nuclease exposure and solving the problems of stability and leakage.

[0010] Three is the problem of single endogenous stimulus response: Existing stimulus-responsive systems mostly rely on exogenous triggers (such as light / heat) or have insufficient recognition ability for single endogenous markers. APNS uses overexpressed miR-21 as an endogenous bifunctional trigger (releasing siPLK1 / Dox and activating the fluorescence signal simultaneously) to achieve precise spatio-temporal control of treatment.

[0011] Fourth, there is the problem of insufficient treatment-diagnosis synergy: The traditional diagnosis and treatment system has problems such as low fluorescence signal sensitivity and poor synergy between therapeutic genes and chemotherapeutic drugs. APNS amplifies the fluorescence signal triggered by miR-21 (diagnostic enhancement) through the Fuel chain competitive displacement mechanism. At the same time, miRNA triggers the synchronous release of siPLK1 (gene therapy) and Dox (chemotherapy), realizing the integration of diagnosis and treatment and synergistically enhancing the anti-tumor efficacy.

[0012] Fifth, there is the problem of limited nucleic acid drug delivery efficiency and gene silencing effect: The existing gene delivery systems have poor gene silencing effects due to the low intracellular release efficiency of siRNA. APNS triggers the in-situ release of siPLK1 through miR-21, combined with the enhanced endocytosis mediated by nucleic acid aptamers, ensuring the efficient release of siPLK1 in the target cell cytoplasm and improving the PLK1 gene silencing efficiency.

[0013] Sixth, there is the problem of insufficient integration of multimodal therapy: It is difficult for the existing technologies to integrate targeting, imaging, gene therapy, and chemotherapy in the same nanoplatform. APNS realizes the integration of four functions through a functionalized core-shell structure: nucleic acid aptamer targeting (AS1411), fluorescence imaging (Fuel chain displacement signal amplification), gene therapy (siPLK1), and chemotherapy (Dox), breaking through the limitations of a single treatment mode.

[0014] Seventh, there is the problem of the lack of real-time monitoring and dynamic feedback: The traditional treatment system lacks visual monitoring during the treatment process. APNS realizes the visual tracking (imaging guidance) of the treatment process through the fluorescence signal activation mechanism triggered by miR-21, providing real-time feedback for efficacy evaluation. Summary of the Invention

[0015] In view of the defects of the existing technologies, through research, the present invention reduces the siRNA degradation rate to less than 5% of that of traditional carriers in the dense reticular DNA outer shell layer formed by the three-dimensional self-assembly of Y-shaped DNA scaffolds, and improves the stability by more than 25 times in a serum environment; the lipid micelle-DNA composite core-shell structure achieves a drug encapsulation rate > 95%, among which the Dox loading reaches 89.5 w / w, and the siRNA binding efficiency is about 100%. Thus, the present invention is completed.

[0016] The present invention provides a core-shell protected intelligent lipid delivery system, and its structure is: The Y-shaped DNA scaffold forms a dense reticular DNA outer shell through cross-linking with double-stranded DNA, namely L-shaped dsDNA, and further hybridizes with the DNA on the surface of cholesterol micelles to be anchored on the surface of cholesterol micelles to form a stable core-shell structure, and a targeted core-shell system is constructed by modifying nucleic acid aptamers on the outer shell surface.

[0017] Specifically, it is obtained by fully mixing and incubating a functionalized lipid micelle core, a Y-shaped DNA scaffold, L-shaped dsDNA, and a nucleic acid aptamer corresponding to the target, ultimately obtaining a core-shell protected intelligent lipid delivery system.

[0018] More specifically, the Y-shaped DNA scaffold is composed of Y1 with the nucleotide sequence shown in SEQ ID No. 5, Y2 with the nucleotide sequence shown in SEQ ID No. 6, and Y3 with the nucleotide sequence shown in SEQ ID No. 7; Among them, the nucleic acid aptamer is, for example, AS1411 with the nucleotide sequence shown in SEQ ID No. 10.

[0019] Preferably, the L-shaped double-stranded DNA is composed of L1 with the nucleotide sequence shown in SEQ ID No. 8 and L2 with the nucleotide sequence shown in SEQ ID No. 9.

[0020] In a specific embodiment, the functionalized lipid micelle core is obtained by mixing and incubating a Fuel DNA strand, a pre-formed cholesterol micelle, and a D1 / Linker complex; Specifically, the nucleotide sequence of the Fuel DNA strand is as shown in SEQ ID No. 4, and a cholesterol micelle is pre-formed by the hydrophobic aggregation of Chol-DNA. The nucleotide sequence of Chol-DNA is as shown in SEQ ID No. 1; the D1 / Linker complex is formed by base complementary pairing between D1 and Linker. The nucleotide sequences of D1 and Linker are shown in SEQ ID No. 2 and SEQ ID No. 3 respectively, and D1 is modified, for example, modified with a fluorescent label, for example, modified with BHQ1; The present invention also provides a preparation method of the core-shell protected intelligent lipid delivery system, which is characterized by including the following steps: (1) Construction of the functionalized lipid micelle core: Mix the Fuel DNA strand, the pre-formed cholesterol micelle, and the D1 / Linker complex, centrifuge and then incubate to obtain the functionalized lipid micelle core; (2) Assembly of the Y-shaped DNA scaffold: Take equimolar amounts of Y1, Y2, and Y3, add them to a buffer and mix well, heat at 90 - 100 °C (for example, 95 °C) and then slowly cool to room temperature to form a Y-shaped three-dimensional scaffold; (3) Preparation of the L-shaped dsDNA: Take equimolar amounts of L1 and L2, add them to a buffer and mix well, heat, and then slowly cool to room temperature to obtain the L-shaped dsDNA structure; (4) APNS self-assembly: Mix the above functionalized lipid micelle core, Y-shaped DNA scaffold, L-shaped dsDNA, and nucleic acid aptamer thoroughly (preferably at a molar ratio of 1:1:1.5:1), and incubate to obtain a core-shell protected intelligent lipid delivery system.

[0021] Specifically, in step (1), the Fuel DNA strand, pre-formed cholesterol micelles, and D1 / Linker complex are mixed at a molar ratio of 1:3:1; The preparation method of the pre-formed cholesterol micelles is as follows: Take Chol-DNA, add buffer and mix well, heat, and then slowly cool to room temperature to obtain cholesterol micelles; In step (2), the heating melting temperature is 95 °C; in step (4), the functionalized lipid micelle core, Y-shaped DNA scaffold, L-shaped dsDNA, and nucleic acid aptamer are mixed at a molar ratio of 1:1:1.5:1.

[0022] The preparation method of the D1 / Linker complex is as follows: Take equimolar amounts of D1 and Linker, add buffer and mix well, heat, and then slowly cool to room temperature to obtain the D1 / Linker complex.

[0023] The present invention also provides the application of the core-shell protected intelligent lipid delivery system in delivering drugs, functional substances, or active substances.

[0024] Specifically, it is used for any of the following purposes: (1) For delivering chemotherapeutic drugs, such as for the combined treatment of tumor chemotherapy and gene therapy; (2) For targeted delivery to reduce systemic toxicity. For example, by introducing the nucleic acid aptamer AS1411 on the Y-shaped DNA scaffold, it can bind to nucleolin highly expressed on the surface of therapeutic cells, guide APNS to accumulate in tumor tissues, reduce damage to normal tissues, especially reduce the toxic and side effects on organs such as the heart and liver. It is applicable to patients with metastatic cancer, hematological tumors (such as leukemia), and those who need to reduce chemotherapy toxicity; (3) For an intelligent response-type release system, that is, a drug controlled-release response safety switch system inside the core-shell protected intelligent lipid delivery system; For example, a specific miRNA (such as tumor-related miR-21, miR-10b) response-type drug delivery system to achieve "intelligent" controlled release; (4) For simultaneously delivering chemotherapeutic drugs, drug resistance reversers (such as P-glycoprotein inhibitors), or pro-apoptotic siRNAs to reverse tumor drug resistance; (5) For immune combination therapy, i.e., delivering combined chemotherapy and immunomodulation; delivering immune checkpoint inhibitors (such as PD-1 siRNA) or immune activators (such as oligonucleotides like CpG, ASO, etc.), and synergistically activating anti-tumor immunity with chemotherapy. (6) For theranostics, i.e., combining treatment and real-time monitoring, integrating imaging agents (such as fluorescent probes, MRI contrast agents) with therapeutic drugs to achieve synchronous treatment and efficacy monitoring. Modifying near-infrared fluorescent groups or superparamagnetic iron oxide (SPIO) through a DNA scaffold, and encapsulating drugs with lipid micelles. Tracking the drug distribution and tumor regression through imaging techniques (such as fluorescence imaging, nuclear magnetic resonance imaging), which is applicable to intraoperative navigation, dynamic evaluation of treatment efficacy, and personalized dose adjustment. (7) For delivering anti-inflammatory drugs (such as dexamethasone) and siRNA targeting inflammatory factors (such as TNF-α siRNA), which is applicable to rheumatoid arthritis or inflammatory bowel disease.

[0025] (8) For neurodegenerative diseases: Crossing the blood-brain barrier to deliver neurotrophic factors or gene editing tools (such as CRISPR-Cas9), which is applicable to the treatment of Alzheimer's disease and Parkinson's disease. Specifically, it is used for dynamic controlled release, including the first stage: miRNA triggers the preferential release of siRNA, which enters the cytoplasm through endosomal escape; the second stage: the release of siRNA triggers the displacement of the Linker and the Fuel chain, further releasing siRNA and simultaneously triggering the release of Dox; the third stage: the core-shell structure disintegrates in the acidic environment of lysosomes to further release Dox, which penetrates into the nucleus.

[0026] Based on the technical solution of the present invention, it can be used in the following aspects to obtain good effects: In terms of the targeted precise delivery system: The tumor cell-specific recognition mediated by the modification of the nucleic acid aptamer AS1411 increases the tumor cell uptake efficiency by 3.92 times (verified by flow cytometry), significantly enhancing the drug enrichment degree at the tumor site (targeting efficiency > 98%); the drug accumulation in normal tissues is reduced to 14.2% of the naked drug group.

[0027] Intelligent responsive drug release mechanism: Specifically responding to the miR-21 overexpression environment to achieve precise drug release triggered by endogenous miRNAs in tumors; constructing a signal amplification circuit through the Fuel chain displacement reaction, significantly enhancing the fluorescence detection sensitivity and at the same time enhancing the RNA interference efficiency.

[0028] In terms of synergistic treatment enhancement: It can be used in chemotherapy-gene therapy synergistic enhancement systems. For example, co-loading Dox and siPLK1 drugs achieves drug synergistic enhancement; the tumor growth inhibition rate is increased by 20 - 40% compared to single-drug treatment. For the aspect of integrated diagnosis and treatment: It is used in fluorescence signal amplification systems. For example, real-time monitoring during the treatment process is achieved through miR-21-responsive fluorescent probes; the fluorescence intensity is enhanced by 3.9 times through Fuel chain competitive displacement (excitation at 535 nm); the detection sensitivity of miR-21 reaches 1 fM, and the linear range spans 6 orders of magnitude; for in-situ gene intervention, for example, the RNAi efficiency is increased by 1.22 times compared to traditional liposomes (qPCR verifies the expression of PLK1 mRNA); the synergistic anti-tumor effect is significantly enhanced.

[0029] In terms of innovation in formulation technology, the standardized ligation efficiency of the Y-shaped DNA scaffold > 95%; the drug loading rate has a breakthrough increase (Dox ≈ 89.5%, siRNA ≈ 100%).

[0030] In terms of the advantages of clinical application transformation, it can be used for the integration of multimodal therapies. For example, the combination of chemotherapy and gene therapy increases the tumor inhibition rate to 86.8% (nude mouse model); the systemic circulation half-life is greatly extended compared to conventional formulations; a closed-loop system for anti-tumor "diagnosis - treatment - efficacy monitoring" is achieved.

[0031] In summary, through innovative designs such as molecular self-assembly engineering, structural innovation and functional integration, bioresponsive intelligent release, and multimodal synergistic therapy, the present invention has overcome key technical bottlenecks such as poor stability of gene drug delivery, weak targeting of chemotherapy drugs, and separation of diagnostic and therapeutic functions, and has important clinical application value in the field of anti-tumor precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To more clearly illustrate the technical solutions and innovation advantages of the embodiments of the present invention, the specific embodiments and experimental data of the present invention will be described below in conjunction with the drawings: Figure 1 Demonstrates the construction mechanism and synergistic treatment principle of the APNS nanocomposite system. Among them, A illustrates the construction strategy of the ternary composite structure of APNS / siPLK1 / Dox through a schematic diagram of the self-assembly process; B reveals the intelligent response mechanism of this nanosystem in the tumor microenvironment, triggering the sequential controlled release of the siPLK1 gene silencer and doxorubicin (Dox) through endogenous miRNA, and achieving the synergistic enhancement of gene therapy and chemotherapy.

[0033] Figure 2 Characterizes the construction of the APNS nanocomposite system, and characterizes the structural integrity and nanoparticle morphology of the APNS system through 3% agarose gel electrophoresis (A) combined with transmission electron microscopy (TEM) (B) to verify its successful assembly.

[0034] Figure 3 The stability of the APNS system (A), the loosely modified lipid micelles with a Y-shaped shell (B), the DNA-functionalized lipid micelle core (C), and naked siPLK1 (D) in a 10% fetal bovine serum environment was evaluated by 12% native polyacrylamide gel electrophoresis, and their ability to resist nuclease degradation was quantitatively analyzed by the nucleic acid retention rate at different time points (0 - 48 h).

[0035] Figure 4 Through laser confocal microscopy imaging and flow cytometry quantitative analysis, the ability of the APNS system to target and recognize specific receptors on the surface of tumor cells (A and B) and normal cells (C and D) was confirmed, demonstrating its active targeting drug delivery characteristics.

[0036] Figure 5 By comprehensively using a fluorescence spectrophotometer (F-7000) (A and D), dynamic light scattering (DLS) (B), and native polyacrylamide gel electrophoresis (C), the drug loading efficiency of the APNS system for siPLK1 and Dox, the dynamic changes in Zeta potential, and the miRNA-responsive release characteristics were systematically studied.

[0037] Figure 6 Through quantitative real-time fluorescence PCR (qRT-PCR) (A) and Western blot (WB) (B) experiments, the inhibitory effects of APNS / siPLK1 on the mRNA and protein expression levels of the PLK1 gene in A549 cells were quantitatively evaluated.

[0038] Figure 7 Based on the CCK-8 method, the cytotoxicity differences of the APNS system against target cells (A549) (A and C) and non-target normal cells (HELF) (B) were detected to verify its selective killing effect.

[0039] Figure 8 By using the Annexin V-FITC / PI double staining method combined with flow cytometry, the biological effect of the APNS system in inducing apoptosis of target tumor cells was quantitatively analyzed to reveal its anti-cancer mechanism of action. Specific implementation manners

[0040] Based on Figure 1 As shown, it demonstrates the engineering design and mechanism of action of a core-shell structured co-delivery nanosystem of siPLK1 and doxorubicin (Dox) (abbreviated as APNS). This intelligent delivery system forms a unique cholesterol-based lipid micelle structure through molecular self-assembly. Its core is formed by the self-aggregation of hydrophobic cholesterol-modified DNA (Chol-DNA), and a dense reticular outer shell is constructed on the surface through multi-level DNA self-assembly. Specifically:[[]]END]] 1. Core construction: Chol-DNA, as an amphiphilic structural unit, consists of three functional regions: (i) a hydrophilic DNA segment; (ii) a hydrophobic cholesterol group; (iii) a TEG linker. Self-assembly is driven by hydrophobic interactions to form a micelle core, and the D1 / Linker complex and Fuel DNA strand are hybridized with the hydrophilic DNA in the lipid micelle through base complementary pairing and anchored on the micelle surface.

[0041] 2. Functionalized shell construction: A stepwise self-assembly strategy is adopted: (i) Y-Apt specifically binds to double-stranded DNA (dsDNA) through two terminal sequences; (ii) dsDNA serves as a cross-linker to connect Y-Apt units to form a three-dimensional network structure. This dense shell co-packages siPLK1 and Dox within the core-shell structure through physical confinement, providing dual protection, a nuclease degradation resistance barrier and a drug leakage barrier.

[0042] 3. Intelligent response mechanism, including 1) Construction of a dual-signal-triggered cascade release system: Fluorescence reporting system: The 5' end of siPLK1 is modified with a Cy3 fluorophore, and the 3' end of D1 is modified with a BHQ1 quenching group. D1 and siPLK1 form a fluorescence resonance energy transfer (FRET) pair by hybridizing with the Linker strand to monitor drug release in real time.

[0043] Cascade strand displacement reaction: Temporal control release is achieved through a toehold-mediated sequence competition mechanism: Endogenous miRNA initiates the first round of displacement through toehold a, releasing siPLK1 (fluorescence recovery); the exposed toehold b triggers the second round of displacement driven by FuelDNA, releasing Dox and regenerating miRNA; the released miRNA participates in the next cycle to achieve a signal amplification effect.

[0044] 4. System optimization characteristics 1) Targeting enhancement: Y-Apt is modified with the nucleic acid aptamer AS1411, and active targeting mediated by the nucleolin receptor increases tumor accumulation; 2) Stability optimization: The cholesterol micelle core and the dense DNA shell cooperate to enhance nuclease resistance; 3) Reaction kinetics enhancement: The steric hindrance effect generated by the high-density DNA arrangement on the surface reduces non-specific binding and improves the strand displacement efficiency.

[0045] The innovative design of the present invention realizes the synergistic enhancement of chemotherapy-gene therapy through spatiotemporally controllable drug release, self-feedback fluorescence monitoring, and a cascade amplification drug release mechanism, providing a new nanotechnology platform for anti-tumor combination therapy.

[0046] Example 1: Construction and Characterization of APNS System To verify the assembly of APNS, this study characterized the system by agarose gel electrophoresis (3% AGE) and transmission electron microscopy (TEM). The specific experimental methods are as follows: 1. Preparation of Core-Shell Structure APNS (1) Construction of functionalized lipid micelle core: Mix Fuel DNA strand (1 µL, 10 µM) (SEQ ID No. 4), (3 µL, 10 µM) Chol-DNA (SEQ ID No. 1) pre-formed hydrophobic aggregates of cholesterol micelles, equimolar amounts of D1 (1µL, 10 µM) (SEQ ID No. 2) and Linker (1 µL, 10 µM) (SEQ ID No. 3) pre-hybridized to form D1 / Linker complex, 0.7 µL 10× PBS and 0.3 µL ddH2O in an EP tube by vortexing, centrifuging and incubating at 37 °C for 1 h to obtain the functionalized lipid micelle core; (2) Assembly of Y-shaped DNA scaffold: Take equimolar amounts of Y1 (SEQ ID No. 5), Y2 (SEQ ID No. 6), Y3 (SEQ ID No. 7) (1 µL, 10 µM), add 0.4 µL 10× PBS and 0.6 µL ddH2O and mix well, heat at 95 °C for 5 min, then slowly cool to room temperature to form a Y-shaped three-dimensional scaffold; (3) Preparation of L-shaped dsDNA: Take equimolar amounts of L1 (SEQ ID No. 8), L2 (SEQ ID No. 9) (1.5 µL, 10 µM), add 0.4 µL 10× PBS and 0.6 µL ddH2O and mix well, heat at 95 °C for 5 min, then slowly cool to room temperature to obtain the L-shaped dsDNA structure; (4) Self-assembly of APNS: Mix the above functionalized lipid micelle core (7 µL), Y-shaped DNA scaffold (4 µL), L-shaped dsDNA (4 µL) and nucleic acid aptamer AS1411 (SEQ ID No. 10) (1 µL, 10 µM) thoroughly, incubate at 37 °C for 1 h, and finally obtain the core-shell structure APNS.

[0047] 2. Agarose Gel Electrophoresis Analysis (AGE) (1) Gel preparation: Weigh agarose powder at a concentration of 3% (w / v), dissolve it in 0.5×TBE buffer, heat it in a microwave to dissolve, and pour it into a gel plate with a comb to form a gel; (2)Sample preparation: Take 16 μL of APNS solution and dilute it to 20 μL with 4 μL of ddH2O. Then take 8 μL of the diluted solution and mix it with 4 μL of 6× loading buffer. The Marker group consists of 0.5 μL of DNA Ladder (2000 bp), 4 μL of loading buffer, and 7.5 μL of ddH2O. (3)Electrophoresis conditions: In a 0.5× TBE buffer system, perform electrophoresis at a constant voltage of 110 V for 30 min. After electrophoresis, perform SYBR Green fluorescence imaging using a gel imaging system (ChemDoc XRS, Bio-Rad, USA).

[0048] The results are as Figure 2 shown in A below. Lanes 1 - 4 are lipid micelles, lipid micelle cores, Y-Apt modified micelles, and APNS respectively. Bands with gradually decreasing mobility were observed, and this migration retardation phenomenon effectively confirmed the stepwise construction of APNS.

[0049] 3. Transmission electron microscopy (TEM) characterization Dilute the APNS solution to 250 nM with 1× PBS. Take 10 μL of the diluted solution and drop it onto a carbon-supported copper grid (Holey Carbon Grid, 300 mesh). Let it stand at 37 °C for 30 min. After adsorption, remove the residual liquid with a filter paper. After secondary drying, place it in the sample chamber of a transmission electron microscope (TEM, 200 kV, JEM-F200). Collect bright-field images at an acceleration voltage of 200 kV and perform particle size statistical analysis using Gatan MicroscopySuite software.

[0050] As shown by TEM characterization ( Figure 2 in B below), APNS presents a spherical nanostructure with good monodispersity, and its average particle size is 191.0 ± 18.90 nm, which meets the ideal size distribution of nanodrug carriers.

[0051] Table 1. Sequences used in each experimental group

[0052] Example 2. Evaluation of the stability of the APNS system against antiserum Since drug delivery systems will inevitably come into contact with complex biological media such as nucleases and serum in vivo, the anti-degradation ability of the carrier is a key indicator to ensure the effectiveness of the drug. In this study, 10% fetal bovine serum (FBS) was used to simulate the physiological environment, and the nuclease resistance of the core-shell structured APNS and its control carriers was systematically evaluated.

[0053] The experimental design is as follows: The APNS prepared in Example 1, the lipid micelles loosely modified with a Y-shaped outer shell, the DNA-functionalized lipid micelle core, and naked siPLK1 (the nucleic acid sequence of the siPLK1 is as follows: sense strand 5'-(Cy3)CCC UAU AGCUUA UCA GAC U UG AAG AAG AUC ACC CUC CUU A dTdT-3' (SEQ ID No. 11); antisense strand 5'- UAA GGA GGG UGA UCU UCU UCA dTdT-3' (SEQ ID No. 12)) were respectively mixed with 10% FBS and incubated at 37 °C for 0, 2, 4, 6, 8, 12, 24, and 48 h. After terminating the reaction, the products were analyzed by 12% native polyacrylamide gel electrophoresis (n-PAGE), and band quantification was performed with Image Lab software. Finally, data visualization was carried out using Origin.

[0054] As Figure 3 shown, the degradation kinetics of each carrier showed significant differences: The lipid micelles loosely modified with a Y-shaped outer shell had a retention rate of 89.67% after 48 h, indicating insufficient protection of its outer shell structure against nuclease attack; the degradation of the DNA-functionalized lipid micelle core was more obvious, with only 60.33% remaining after 48 h; naked siPLK1 underwent rapid degradation within 2 h, and the electrophoresis band became significantly lighter. In contrast, the core-shell structured APNS showed excellent stability, and no obvious degradation of siPLK1 was observed after 48 h of incubation.

[0055] The above results indicate that the core-shell structure formed by the tight coupling of the Y-shaped DNA outer shell and the lipid micelles can effectively block the attack of nucleases in serum on the inner core siPLK1. This dual protection mechanism combining steric hindrance and chemical stability significantly improves the stability of nucleic acid drugs in complex biological environments and lays an important foundation for subsequent clinical applications.

[0056] Example 3. Study on the targeting recognition ability of the APNS system Traditional chemotherapeutic drugs often suffer from limited systemic toxicity and efficacy due to lack of targeting. The nucleic acid aptamer-functionalized nanosystem (APNS) prepared in Example 1 of this study achieves specific targeting through the self-assembly of the nucleic acid aptamer AS1411. Nucleolin (NCL), as a tumor-specific marker, is highly expressed in A549 human non-small cell lung cancer cells and lowly expressed in HELF human embryonic lung fibroblasts. Based on this biological characteristic, we selected A549 as the target cell and HELF as the non-target control cell for targeting verification.

[0057] The specific experimental method is as follows: 1. Laser confocal microscopy analysis: Cells in the logarithmic growth phase were digested with 0.25% trypsin and seeded at 1×10 5 cells / well in a 24-well plate pre-coated with glass slides. After mixing by the cross method, the cells were cultured at 37 °C and 5% CO2 for 24 h. After removing the medium, the cells were washed 3 times with PBS, and then 500 μL of serum-free DMEM containing 40 nM Cy5-APNS was added. The cells were incubated in the dark for 2 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 min and stained with DAPI for 30 min. After mounting with an anti-quenching agent, dual-channel imaging was performed using a laser confocal microscope (CLSM, Nikon A1R MP, Japan). The excitation wavelengths were Cy5 640 nm / DAPI 405 nm.

[0058] After co-incubating cells with APNS labeled with Cy5 fluorescence for 2 hours, laser confocal microscopy imaging showed ( Figure 4 A, C in), significant Cy5 red fluorescence signals were presented in the cytoplasm of A549 cells, while the fluorescence signals in HELF cells were almost absent, confirming that the modification of AS1411 could endow APNS with precise targeting ability.

[0059] 2. Flow cytometry quantification: Cells were seeded at 2×10 5 cells / well in a 6-well plate. After mixing by the cross method, the cells were cultured at 37 °C and 5% CO2 for 24 h. After removing the medium, the cells were washed 3 times with PBS, and then 500 μL of serum-free DMEM containing 40 nM Cy5-APNS was added. The cells were incubated in the dark for 2 h. After digestion with trypsin, a single-cell suspension was prepared, washed 3 times with PBS, and filtered through a 300-mesh nylon membrane. Detection was performed using a CytoFLEX flow cytometer (Beckman Coulter). 10,000 effective cell signals were collected in the Cy5 channel, and fluorescence intensity statistical analysis was performed using FlowJo v10 software.

[0060] Flow cytometry quantitative analysis further verified this result ( Figure 4 B, D in), which was highly consistent with the conclusion of microscopic observation.

[0061] Example 4. Study on the co-loading and stimulus-responsive release of siPLK1 and Dox by the APNS system Doxorubicin (Dox), a widely used chemotherapeutic drug for tumors in clinical practice, can inhibit nucleic acid synthesis by inserting into the G-C base pairs of the DNA double helix structure, thereby inducing apoptosis and autophagy of tumor cells. In this study, the drug-loading research was carried out using the unique fluorescence tunable property of Dox: free Dox exhibits double characteristic emission peaks at 560 nm and 590 nm under 488 nm excitation, while the signal disappears due to the fluorescence quenching effect when inserted into the DNA double strand. Based on this principle, we first systematically evaluated the Dox-loading capacity of APNS by fluorescence spectroscopy. As Figure 5 shown in A, after co-incubating gradient concentrations of APNS (0 - 100 nM) with 2 μM Dox for 12 h, it was found that as the concentration of APNS increased, the fluorescence intensity of Dox decreased in a concentration-dependent manner, indicating that Dox was successfully inserted into the DNA structure of APNS. When the concentration of APNS reached 50 nM, the fluorescence intensity reached a plateau, suggesting that Dox was fully loaded at this time. Based on this result, 50 nM APNS and 2 μM Dox were selected for subsequent experiments to construct a drug-loaded complex (APNS / Dox).

[0062] To verify the co-loading efficiency of siPLK1 and Dox, we further characterized the surface charge change of the system by zeta potential analysis ( Figure 5 shown in B). The results showed that naked siPLK1 and free Dox presented zeta potentials of -6.7 ± 1.2 mV and 6.6 ± 1.0 mV, respectively, while pure APNS carried a strong negative charge (-42.2 ± 4.7 mV) due to the backbone phosphate groups. When siPLK1 was loaded, the potential of the APNS / siPLK1 complex decreased significantly to -48.5 ± 4.1 mV, and this change was due to the hybridization binding of negatively charged siPLK1 to APNS. Notably, the potential of the APNS / siPLK1 / Dox system recovered to -41.5 ± 3.5 mV, which was attributed to the neutralization of part of the surface negative charge by the positively charged Dox molecules, confirming the co-loading of siPLK1 and Dox.

[0063] To achieve precise controlled release of the drug, we introduced a miRNA-responsive element (using miRNA-21 as a proof of concept, and the nucleic acid sequence of the miRNA-21 is as follows: 5’-TAG CTT ATC AGA CTG ATG TTG A-3’ (SEQ ID No. 13)) into the APNS structure. When the complex enters tumor cells, endogenous miRNA can trigger a strand displacement reaction through base complementary pairing, promoting the release of siPLK1 ( Figure 5In C). Agarose gel electrophoresis verified this release mechanism: compared with pure APNS / siPLK1 (lane 3), obvious characteristic bands of siPLK1 appeared in the miRNA treatment group (lane 4). Through real-time fluorescence monitoring, it was found that under the stimulation of 25 nM miRNA, the fluorescence signals of siPLK1 (Cy3-labeled) and Dox both increased with the extension of time ( Figure 5 In D), which confirmed that miRNA could not only trigger the release of siPLK1, but also promote the de-embedding of Dox through the subsequent hybridization reaction between Fuel DNA and Linker, realizing the sequential controlled release of the two drugs.

[0064] Example 5. Inhibition of PLK1 gene by APNS system loaded with siPLK1 To evaluate the gene silencing efficacy of the APNS / siPLK1 complex, after verifying the targeted delivery characteristics of the APNS system by laser confocal microscopy in this study, A549 cells were co-incubated with APNS / siPLK1 (equivalent concentration of 120 nM), free siPLK1 and empty APNS for 48 hours, and then the inhibitory effects of the PLK1 gene on the mRNA and protein levels were detected by qRT-PCR and Western blotting techniques respectively.

[0065] 1. Analysis of gene expression levels Experimental group setting: A549 cells were co-incubated with APNS / siPLK1 (120 nM), naked siPLK1, empty APNS and PBS control for 48 hours. qRT-PCR detection showed ( Figure 6 A) that the expression level of PLK1 mRNA in the APNS / siPLK1 treatment group decreased significantly to 18.47% of the control group, while free siPLK1 and the empty group did not show significant inhibitory effects. This phenomenon was attributed to the dual protection mechanism of the APNS core-shell structure on siRNA: 1) The inner core stably encapsulated siPLK1 through base complementary pairing hybridization, effectively resisting nuclease degradation; 2) The active targeting mediated by the surface AS1411 nucleic acid aptamer significantly improved the internalization efficiency of nanoparticles in A549 cells.

[0066] 2. Verification of protein expression Western Blot analysis further confirmed ( Figure 6 B) that the expression level of PLK1 protein in the APNS / siPLK1 treatment group decreased significantly to 21.40% of the control group, which was highly consistent with the mRNA inhibition trend. However, due to the low intracellular delivery efficiency of the naked siPLK1 group, effective gene silencing could not be achieved.

[0067] The specific experimental methods are as follows: 1. Detection by real-time fluorescence quantitative PCR 1) RNA Extraction and Quality Control (1)Cell treatment: A549 cells in the logarithmic growth phase (2×10 5 / well) were seeded in 6-well plates, mixed evenly by the cross method, and cultured at 37 °C and 5% CO2 for 24 h. After removing the medium, the cells were washed 3 times with PBS, and 500 μL of serum-free DMEM medium containing different treatment groups was added respectively, and incubated at 37 °C / 5% CO2 for 48 h; (2)Total RNA was extracted by the Trizol method. After gradient purification with chloroform-isopropanol, the purity was evaluated using Nanodrop 2000 (A260 / A280 = 1.92 ± 0.05); 2) cDNA Synthesis System The following mixture was prepared in a 200 µL enzyme-free EP tube:

[0068] Reaction program: 50 °C for 15 min → 85 °C for 5 s → Store at 4 °C. If not used for a long time, store at -30 °C; avoid repeated freezing and thawing.

[0069] 3) qPCR Amplification (1)The required qPCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., dissolved in ddH2O, and stored at -30 °C for later use;

[0070] (2)The qPCR reaction system was prepared according to the following table and aliquoted into eight-strip tubes:

[0071] (3)After mixing the eight-strip tubes evenly, centrifuge to avoid generating bubbles, and load onto the machine for qPCR. The reaction program is as follows:

[0072] (4)Data analysis: After the qPCR program ended, the eight-strip tubes were taken out, the data was exported from Bio-Rad, and analyzed by the ΔΔCt method.

[0073] 2. Western Blot (WB) 1) Protein Extraction and Quantification (1)Cell culture: A549 cells in the logarithmic growth phase were digested and mixed evenly, and then seeded at 2×10 per well 5Plate cells in a 6-well plate, place the cell culture plate in an incubator at 37°C with 5% CO2 for 24 h. Discard the old medium, wash 2-3 times with sterile PBS, add 500 µL of PBS, siPLK1, APNS, and APNS / siPLK1 diluted with serum-free DMEM medium respectively, and place in the cell culture incubator at 37°C with 5% CO2 for incubation for 48 h; (2)Extract total cellular proteins: Pre-cool a high-speed centrifuge to 4°C in advance. Prepare protease lysis buffer by mixing RIPA lysis buffer and protease inhibitor at a ratio of 100:1, and place it on ice for pre-cooling. Take out the 6-well plate, discard the old medium, add 1 mL of PBS to wash 2-3 times, add 60 µL of pre-cooled protease lysis buffer to each well, gently shake until the liquid covers the bottom of the plate, and lyse on ice for 5 min. Then use a clean cell scraper to scrape the cells off, collect the viscous cell lysate into a 1.5 mL EP tube, centrifuge at 12,000 rpm at 4°C for 30 min, and collect the supernatant into a new 1.5 mL EP tube and label it; (3)Protein concentration determination (BCA method): Take a 96-well plate, add 0, 1, 2, 4, 8, 12, 16, 20 µL of protein standard (0.5 mg / mL) and 20, 19, 18, 16, 12, 8, 4, and 0 µL of PBS to the protein standard wells respectively; add 2 µL of the protein solution to be measured and 18 µL of PBS to the sample wells respectively. Finally, add 200 µL of BCA working solution prepared in advance at a ratio of A solution:B solution = 50:1 to each well, mix gently to avoid generating bubbles, then place in a 37°C incubator for incubation in the dark for 30 min, and finally measure the absorbance value at 562 nm of each well with a microplate reader. The correlation coefficient (R 2 ) should be greater than 0.99, and the concentration of the protein sample to be measured can be calculated according to the standard curve; (4)Protein denaturation: Dilute each protein sample with RIPA lysis buffer and 5× Loading buffer to 1 mg / mL, mix well, place in a 100°C constant temperature metal bath for denaturation for 10 min, take out and store at -30°C.

[0074] 2)SDS-PAGE electrophoresis: (1)Gel preparation: Take thin and thick plates with a thickness of 1.5 mm, wipe them clean with absolute ethanol, align them, and firmly clamp them on the gel preparation rack. Prepare the separating gel according to the formula in (2) of 2.2.2.2. After mixing, use a Pasteur pipette to pour the gel to about 2 / 3 of the height, slowly add ddH2O to flatten the separating gel. After the separating gel naturally solidifies for 30 min, an obvious boundary line can be observed. At this time, pour out the ddH2O and blot the residual moisture with absorbent paper; Prepare the stacking gel according to the formula in (3) of 2.2.2.2. After mixing, use a Pasteur pipette to pour the gel, gently insert a comb with a thickness of 1.5 mm, avoid generating bubbles, and let the stacking gel naturally solidify for 30 min; (2)Preparation before electrophoresis: After the stacking gel solidifies, pull out the comb vertically upward, clamp the solidified SDS-PAGE gel with an electrophoresis clip, add the newly prepared 1× electrophoresis buffer in the inner tank, check for any leakage, and then place the non-leaking clamp in the electrophoresis tank. Add 1× electrophoresis buffer to the outer tank to the 1 / 2 position (recycled liquid can be used); (3)Loading: Prepare the protein Marker by mixing 4 µL Marker + 16 µL 1× Loading buffer per well. After centrifuging the protein Marker and each denatured protein sample separately, load 15 µL per lane, and add the protein Marker to both sides; (4)Electrophoresis: Constant voltage of 70 V for 30 min for the stacking gel; 110 V for 70 min for the separating gel; (5)Transfer: Prepare the transfer buffer in advance according to the formula in (2) of 2.2.2.1 and pre-cool it at 4℃. Cut off a corner from the upper right of the PVDF membrane, activate it with methanol for 2 min, and then place it in the transfer buffer for standby. Pour an appropriate amount of pre-cooled transfer buffer into the tray. Place the black side of the transfer clamp downwards, and sequentially place the sponge and filter paper. Gently drive away the bubbles with a test tube. Cut off the stacking gel and the excess gel at the bottom of the gel, and cut off a corner from the upper right. Place the gel flat on the filter paper, place the PVDF membrane with the cut-off right corner flat in the same direction as the gel, avoid generating bubbles, cover with filter paper and sponge, and gently drive away the bubbles with a test tube. Finally, carefully fasten the transfer clamp, place it black side to black in the transfer tank, place an ice pack in the transfer tank, pour the transfer buffer into the tank, cover the lid, place the transfer device in crushed ice, constant current of 300 mA, transfer for 80 min; (6)Blocking: Place the transferred PVDF membrane in the rapid blocking solution and place it on a shaker to slowly block for about 15 min; (7)Primary antibody incubation: Take out the blocked PVDF membrane, put it into 1×TBST, and wash it quickly on a shaker for 3 times, 5 minutes each time. Mark the protein information and position according to the band positions of the protein Marker, the molecular weights of the target protein and the internal reference. Cut out the target protein and internal reference bands, dry the moisture on the filter paper, and then put the protein bands into the corresponding antibody tubes according to the back-to-back principle, and incubate them slowly on a shaker at 4°C for 12 - 16 h; (8)Secondary antibody incubation: Take out the protein bands, put them into 1×TBST, wash them quickly on a shaker for 3 times, 5 minutes each time, dry the moisture with filter paper, and then put them into the secondary antibody tube and incubate them slowly on a shaker for 1.5 h; (9)Exposure: Take out the protein bands, put them into 1×TBST, wash them quickly on a shaker for 3 times, 5 minutes each time. Prepare the exposure solution in advance according to A solution:B solution = 1:1. Take out the protein bands, dry the moisture on the filter paper, evenly cover the surface of the bands with the exposure solution, put them into the gel imager, select the protein exposure program for development, and save the pictures for band quantitative analysis through Image Lab software.

[0075] Example 6. Cytotoxicity evaluation of APNS / siPLK1 / Dox To verify the feasibility of the APNS / siPLK1 / Dox complex as a targeted drug delivery system, this study systematically evaluated the biocompatibility of the material and its specific killing effect on tumor cells by the CCK-8 method. A549 human lung adenocarcinoma cells (PLK1 high-expression tumor model) and HELF human normal lung fibroblasts were selected for comparative analysis.

[0076] 1. Cytotoxicity evaluation of APNS / Dox As Figure 7 shown in A - B below, free doxorubicin (Free Dox) shows dose-dependent cytotoxicity to both types of cells due to lack of targeting. While the APNS material group still maintains a cell survival rate of >95% at a high concentration of 100 nM, confirming its excellent biocompatibility. It is worth noting that the APNS / Dox group shows significantly enhanced cytotoxicity to A549 cells, and the inhibition rate on HELF cells is always <10%. This is attributed to the fact that the AS1411 aptamer modified on the surface of APNS can specifically recognize the overexpressed nucleolin receptor on the tumor cell membrane, realizing targeted drug delivery.

[0077] 2. Verification of synergistic therapeutic effect As Figure 7As shown in C, due to the negative charge repulsion effect and nuclease degradation, naked siPLK1 failed to effectively inhibit the activity of A549 cells (cell survival rate: 98.2%). After loading with APNS, APNS / siPLK1 achieved effective delivery of siRNA through receptor-mediated endocytosis, significantly downregulating the expression of PLK1 protein (see Example 5 for Western blot data), and reducing the cell survival rate to 43.53%. The co-loaded system APNS / siPLK1 / Dox exhibited a significant synergistic effect, with the cell survival rate decreasing to 12.11%, which was 72.2% and 40.3% lower than that of the single-drug treatment groups (APNS / siPLK1: 43.53%; APNS / Dox: 20.29%), respectively.

[0078] 3. Research on apoptosis induction mechanism The cell death mechanism was further analyzed by Annexin V-FITC / PI double staining combined with flow cytometry ( Figure 8 ). The results showed that: (1) The apoptosis rates of the blank control group (PBS) and the naked siPLK1 group were both < 5%; (2) APNS / siPLK1 induced apoptosis in 17.3% of the cells through gene silencing; (3) APNS / Dox caused apoptosis in 29.9% of the cells through chemotherapy; (4) The co-loaded system APNS / siPLK1 / Dox induced apoptosis in 49.6% of the cells, confirming the synergistic effect of gene therapy and chemotherapy; The specific experimental methods are as follows: 1. Cytotoxicity evaluation 1) Toxicity detection of APNS / Dox Take the cell suspension in the logarithmic growth phase (5×10 3 cells / well) and inoculate it into a 96-well plate. After culturing at 37°C and 5% CO2 for 24 h, add the following respectively: (1) Free Dox group: Serum-free DMEM containing 0 - 4 μM Dox; (2) APNS / Dox group: Complex solution with the same drug loading; (3) APNS control group: Blank carrier with the same concentration; After culturing in the dark for 48 h, replace it with the CCK-8 working solution (serum-free DMEM:CCK-8 = 10:1), continue to incubate for 1 - 4 h until the color development is stable, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader (reference wavelength 650 nm).

[0079] 2) Toxicity detection of combined drug administration The cell seeding was the same as before. The experimental groups included: PBS control group, free Dox group (2 μM), naked siPLK1 group (50 nM), APNS / siPLK1 group (siPLK1 50 nM), APNS / Dox group (Dox 2 μM), and APNS / siPLK1 / Dox co-loading group (siRNA 50 nM + Dox 2 μM).

[0080] After culturing in the dark for 48 h, the CCK-8 working solution (serum-free DMEM:CCK-8 = 10:1) was replaced, and incubation was continued for 1 - 4 h until the color development was stable. The absorbance at 450 nm was measured using a microplate reader (reference wavelength 650 nm).

[0081] 2. Detection of cell apoptosis 1) Sample treatment: 1×10 6 cells / well were seeded in a 6-well plate. After culturing for 24 h, the solutions of each treatment group (500 μL / well) were added respectively. After culturing for 48 h, the cells were collected and centrifuged at 4°C and 1500 rpm for 5 min to obtain cell pellets.

[0082] 2) Flow cytometry parameter calibration: 3×10 6 untreated cells were taken, and an apoptosis inducer was added and incubated for 30 min to prepare a positive control. For the single-staining tubes, Annexin V-FITC (5 μL) and PI (10 μL) were added respectively.

[0083] Using a CytoFLEX flow cytometer (Beckman Coulter), detection was performed through the FITC (Ex = 488 nm / Em = 530 nm) and PI (Ex = 535 nm / Em = 617 nm) channels, and the voltage parameters were adjusted according to the blank control.

[0084] 3) Sample detection: After the cell pellets were washed with pre-cooled PBS, they were resuspended in 500 μL of binding buffer. Annexin V-FITC and PI staining solutions were added successively, and after incubation in the dark for 5 min, they were filtered through a 300-mesh nylon membrane and loaded onto the machine. Data analysis was performed using FlowJo V10 software, and the total apoptosis rate was calculated as Q2 (late apoptosis) + Q3 (early apoptosis).

[0085] All technicians should note that although the present invention has been described according to the above specific embodiments, the inventive concept of the present invention is not limited to this invention alone. Any modification using the inventive concept of the present invention will be included in the scope of protection of the patent right of this patent.

[0086] The above is only the preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions within the idea of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A core-shell protective intelligent lipid delivery system, characterized in that, Its structure is as follows: The Y-shaped DNA scaffold crosslinks through double-stranded DNA, namely L-shaped dsDNA, to form a dense network DNA shell, and further hybridizes with the DNA on the surface of the cholesterol micelle to be further anchored on the surface of the cholesterol micelle to form a stable core-shell structure, and a targeted core-shell system is constructed by modifying the nucleic acid aptamer on the surface of the shell.

2. The core-shell protective intelligent lipid delivery system according to claim 1, characterized in that, It is obtained by fully mixing and incubating a functionalized lipid micelle core, a Y-shaped DNA scaffold, L-shaped dsDNA, and a nucleic acid aptamer corresponding to the target, and finally obtaining a core-shell protected intelligent lipid delivery system.

3. The core-shell protective intelligent lipid delivery system according to claim 1, wherein The Y-shaped DNA scaffold is composed of Y1 with the nucleotide sequence shown in SEQ ID No. 5, Y2 with the nucleotide sequence shown in SEQ ID No. 6, and Y3 with the nucleotide sequence shown in SEQ ID No. 7; Specifically, for example, the nucleic acid aptamer is AS1411 with the nucleotide sequence shown in SEQ ID No.

10.

4. The core-shell protective intelligent lipid delivery system according to claim 1, characterized in that The L-shaped double-stranded DNA is composed of L1 with the nucleotide sequence shown in SEQ ID No. 8 and L2 with the nucleotide sequence shown in SEQ ID No.

9.

5. The core-shell protected intelligent lipid delivery system according to claim 1, wherein The functionalized lipid micelle core is obtained by mixing and incubating a Fuel DNA strand, a pre-formed cholesterol micelle, and a D1 / Linker complex; Specifically, the nucleotide sequence of the Fuel DNA strand is as shown in SEQ ID No. 4, and the cholesterol micelle is pre-formed by the hydrophobic aggregation of Chol-DNA, and the nucleotide sequence of Chol-DNA is as shown in SEQ ID No. 1; the D1 / Linker complex is formed by base complementary pairing of D1 and Linker, and the nucleotide sequences of D1 and Linker are shown in SEQ ID No. 2 and SEQ ID No. 3 respectively, and D1 is modified, for example, modified with a fluorescent label, for example, modified with BHQ1.

6. The preparation method of the core-shell protective intelligent lipid delivery system according to any one of claims 1 to 6, characterized in that, It includes the following steps: (1) Construction of the functionalized lipid micelle core: Mix the Fuel DNA strand, the pre-formed cholesterol micelle, and the D1 / Linker complex, centrifuge and then incubate to obtain the functionalized lipid micelle core; (2) Assembly of the Y-shaped DNA scaffold: Take equimolar amounts of Y1, Y2, and Y3, add them to a buffer and mix well, heat at 90-100 °C and then slowly cool to room temperature to form a three-dimensional Y-shaped scaffold; (3) Preparation of L-shaped dsDNA: Take equimolar amounts of L1 and L2, add them to a buffer and mix well, heat, and then slowly cool to room temperature to obtain an L-shaped dsDNA structure; (4) Self-assembly of the functionalized core-shell structure APNS: Fully mix the above-mentioned functionalized lipid micelle core, Y-shaped DNA scaffold, L-shaped dsDNA, and nucleic acid aptamer, and incubate to obtain a core-shell protected intelligent lipid delivery system.

7. The preparation method of the core-shell protective intelligent lipid delivery system according to claim 6, wherein, In step (1), the Fuel DNA strand, the pre-formed cholesterol micelle, and the D1 / Linker complex are mixed in a molar ratio of 1:3:1; The preparation method of preformed cholesterol micelles is as follows: Take Chol-DNA, add buffer and mix evenly, heat, and then slowly cool to room temperature to obtain cholesterol micelles; The preparation method of D1 / Linker complex is as follows: Take equimolar amounts of D1 and Linker, add buffer and mix evenly, heat, and then slowly cool to room temperature to obtain D1 / Linker complex.

8. The preparation method of the core-shell protected intelligent lipid delivery system according to claim 6, wherein, In step (2), the heating melting temperature is 95 °C; in step (4), the functionalized lipid micelle core, Y-shaped DNA scaffold, L-shaped dsDNA, and aptamer are mixed in a molar ratio of 1:1:1.5:

1.

9. The application of the core-shell protective intelligent lipid delivery system according to any one of claims 1 to 5 in delivering drugs, functional substances, or active substances.

10. The application according to claim 9, characterized in that, It is used for any of the following purposes: (1) For delivering chemotherapeutic drugs, such as for the combined treatment of tumor chemotherapy and gene therapy; (2) For targeted delivery to reduce systemic toxicity. For example, by introducing aptamer AS1411 on the Y-shaped DNA scaffold, it can bind to nucleolin highly expressed on the surface of therapeutic cells, guide APNS to enrich in tumor tissues, reduce damage to normal tissues, especially reduce the toxic and side effects on organs such as the heart and liver. Suitable for patients with metastatic cancer, hematological malignancies (such as leukemia), and those who need to reduce chemotherapy toxicity; (3) For an intelligent response release system, that is, a drug-controlled release response safety switch system inside the core-shell protective intelligent lipid delivery system; For example, a specific miRNA (such as tumor-related miR-21, miR-10b)-responsive drug delivery system to achieve "intelligent" controlled release; (4) For simultaneously delivering chemotherapeutic drugs, drug resistance reversal agents (such as P-glycoprotein inhibitors), or pro-apoptotic siRNA to reverse tumor drug resistance; (5) For synergistic immunotherapy, that is, delivering combined chemotherapy and immunomodulation; delivering immune checkpoint inhibitors (such as PD-1 siRNA) or immune activation factors (such as oligonucleotides such as CpG, ASO), and acting synergistically with chemotherapy to activate anti-tumor immune responses; (6) For integrated diagnosis and treatment, that is, combining treatment and real-time monitoring, integrating imaging agents (such as fluorescent probes, MRI contrast agents) with therapeutic drugs to achieve synchronous treatment and efficacy monitoring. By modifying the DNA scaffold with near-infrared fluorescent groups or superparamagnetic iron oxide (SPIO), and loading drugs with lipid micelles. Tracking the drug distribution and tumor regression through imaging techniques (such as fluorescence imaging, magnetic resonance imaging), suitable for intraoperative navigation, dynamic evaluation of treatment efficacy, and personalized dose adjustment; (7) For delivering anti-inflammatory drugs (such as dexamethasone) and siRNA targeting inflammatory factors (such as TNF-α siRNA), suitable for rheumatoid arthritis or inflammatory bowel disease; (8) Neurodegenerative diseases: Crossing the blood-brain barrier to deliver neurotrophic factors or gene editing tools (such as CRISPR-Cas9), suitable for treating Alzheimer's disease and Parkinson's disease; Specifically, it is used for dynamic controlled release, including the first stage: miRNA triggers the preferential release of siRNA, which enters the cytoplasm through endosomal escape; the second stage: the release of siRNA triggers the displacement of the Linker and the Fuel chain, further releasing siRNA and simultaneously triggering the release of Dox; the third stage: the core-shell structure disintegrates in the acidic environment of lysosomes to further release Dox, which penetrates into the nucleus.

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